Ribonucleotidyl terminal transferases have been described from a wide variety of organisms including bacteria [Paine and Boezi, J. Biol. Chem. 248, 4756 (1970); Sippel, Eur. J. Biochem. 37, 31 (1973)], lower eukaryots such as yeast [Haff and Keller, Biochem. Biophys. Res. Comm. 51, 704 (1973)], plants [Mans and Huff, J.Biol. Chem. 250, 3672 (1975); Brishammer and Juntti, Biochem. Biophys. Acta 383, 351 (1975)], and higher animals [Edmonds and Abrams, J. Biol. Chem. 235, 1142 (1960); Cory, et al., Biochem. Biophys. Res. Comm. 42, 778 (1971); Tsiapalis, et al., Biochem. Biophys. Res. Comm. 50, 737 (1973)]. There are riboadenylate terminal transferases specific for AMP addition [Paine and Boezi, J. Biol. Chem. 248, 4756 (1970)], those specific for CMP addition [Edmonds, J. Biol. Chem. 240, 4621 (1965)], those specific for GMP addition [Burkard and Keller, Proc. Nat. Acad. Sci., USA, 71, 389 (1974)], and those specific for UMP addition [Hozumi et al., Nature 256, 337 (1975)]. The riboadenylate terminal transferase activities from the bacteria, Escherichia coli, and from maize plants have been used for 3'-terminal modification of RNA prior to complementary DNA synthesis using reverse transcriptase [Hagenbuechle et al., Cell 13, 551 (1978)]and RNA sequencing [Winter and Brownlee, Nucl. Acids Res. 5, 3129 (1978)].
Ribonucleotidyl terminal transferases are enzymes which modify the 3'-termini of ribonucleic acids (RNA) by the addition of one or more ribonucleotides (CMP, UMP, AMP, or GMP) using ribonucleoside triphosphates (CTP, UTP, ATP, or GTP) as substrates. As mentioned above, such enzymes have been described from a variety of organisms including bacteria, lower eukaryots, plants, and higher animals.
Recent developments in recombinant DNA technology permit the replication and expression of genes from totally different biological classes in particular organisms. With the corresponding discovery of retrovirus reverse transcriptase (RNA-directed DNA polymerase), it is possible to synthesize DNA genes using RNA as template (nucleic acid to be copied) and a primer RNA or DNA molecule preferably hydrogen-bonded (base paired) to the 3'-terminal nucleotides of the RNA template. That some RNAs, specifically eukaryotic cellular messenger RNAs, have a 3'-homopolymer tail of adenylate residues facilitates initiation of synthesis of DNA complementary to the template RNA species at their 3'-termini using a complementary RNA or DNA primer such as oligo(rU) or oligo(dT). However, many other RNA species, of both cellular and viral origin, do not have such a 3'-homopolymer segment. Thus, synthesis of complete DNA gene copies of these RNA species by the above method is not directly possible unless the 3'-termini of such RNA molecules can be modified by the terminal addition of repeating units of a specific ribonucleotide to generate such a 3'-homopolymer tail. The present invention provides enzymes and methods which facilitate such RNA modification, thus making it possible to synthesize in vitro complete DNA copies of RNAs originally void of 3'-homopolymer sequences. Furthermore, one may select for the synthesis of a homopolymer stretch of adenylates, cytidylates, or uridylates.